In many infectious diseases of bacterial origin virulence is mediated by lysogenic conversion of the host bacterium by specific bacteriophages. Of these diseases, diphtheria has been most widely studied. Diphtheria is caused by the bacterium Corynebacterium diphtheriae which is lysogenic for a family of temperate bacteriophages, and the exotoxin that this lysogen expresses. The structural gene for diphtheria toxin, tox is carried by the Corynephage Beta. The ability of a corynebacterial strain to produce toxin depends upon the presence of the phage genome, either as a prophage or as a vegetatively replicating chromosome, and the physiologic state of the host. The expression of the Corynephage Beta tox gene is controlled by host factors and is specifically inhibited by excess iron in the culture medium. My research to date has shown that I can synthesize in good yield diphtheria tox gene products in an E. coli in vitro protein synthesizing system programmed with Corynephage Beta DNA. The expression of tox gene products in the heterologous E. coli protein synthesizing system is insensitive to inhibition by excess iron. The in vitro expression of the tox gene is however inhibited by the addition of C. diphtheriae extracts to the system. The objectives of this proposed research are to make a complete biochemical and genetic analysis of the regulation of the Corynephage Beta tox gene. To that end the experimental plan of this research has five specific goals. These goals are: (a) to develop radioimmune assays for Beta phage gene products synthesized in vitro other than the tox gene; (b) to define the role that iron plays in the inhibition of the tox gene; (c) to purify and characterize the tox gene inhibitory factor from the non-lysogenic, non-toxic C. diphtheriae, C7(-); (d) to define the location and nature of tox gene regulation; and (e) to describe the diphtheria tox operon. The approach to these problems will be made with standard biochemical and genetic techniques.